Rationally designed chromosome fusion does not prevent rapid growth of Vibrio natriegens

DNA replication is essential for the proliferation of all cells. Bacterial chromosomes are replicated bidirectionally from a single origin of replication, with replication proceeding at about 1000 bp per second. For the model organism, Escherichia coli, this translates into a replication time of about 40 min for its 4.6 Mb chromosome. Nevertheless, E. coli can propagate by overlapping replication cycles with a maximum short doubling time of 20 min. The fastest growing bacterium known, Vibrio natriegens, is able to replicate with a generation time of less than 10 min. It has a bipartite genome with chromosome sizes of 3.2 and 1.9 Mb. Is simultaneous replication from two origins a prerequisite for its rapid growth? We fused the two chromosomes of V. natriegens to create a strain carrying one chromosome with a single origin of replication. Compared to the parental, this strain showed no significant deviation in growth rate. This suggests that the split genome is not a prerequisite for rapid growth.

(A) Doubling times are determined to be 22 min 46 s (+/-43.8s) and 23 min 39 s (+/-24.6 s) for the parental and synSC1.0strain, respectively.The difference in growth rate is 4.1%.E. coli was used as a control under the same conditions and doubling time was determined to be 1 h 40 min 12 s (+/-3 min 20.8 s).However, the conditions presumably cause high salt stress for E. coli.Student's t-test was applied to determine the significance; *: p< 0.01, **: p<0.001, n.s.not significant.Experiments were performed biological quadruplicates each with technical triplicates.(B) Growth curve of V. natriegens parental (left panel) and synSC1.0(right panel).We observed no extended lag-phase for synSC1.0 in repeated experiments from dense overnight cultures.Previously, we observed shorter lag-phase for synSC1.0which may be explained by at least a fraction of cells still replication (see Figure S6D-E 2F).It may be the result of reduced chromosome dimer resolution fidelity previously observed in V. cholerae 1 .More severe phenotypes affecting the whole cell population were described earlier for a natural isolate with fused chromosomes of V. cholerae but are not observed for synSC1.0 2 .However, recent literature indicates that there are potential challenges with cell division licensing in MCH1 presumable based on nucleoid occlusion system due to misplacement of the SlmA protein 3,4 .Scale bar 2 µm.

Figure S6 | Coverage plots of samples for replication pattern analysis based on normalized bin read count for each sample.
Normalized marker frequency analysis (1000 bp bins) for (A) the V. natriegens parental strain in exponential growth phase, (B) early stationary phase, (C) the V. natriegens synSC1.0strain in exponential growth phase and (D) in early stationary phase respectively.(E) Visualizes a magnification of (B) and (D) for each replicate indicating that a fraction of V. natriegens synSC1.0 cells in the stationary phase are still replicating DNA.This observation is unexpected because the samples were in regard to their optical density measurements in stationary phase.The samples were taken at a later time point compared to the initial study because this effect was observed earlier 5 .This indicates that the ori:ter ratio of synSC1.0 is higher compared to the plotted value in Figure 3B because in the normalization step where the ratio of replicating/non-replicating data is generated the ratio obtained is reduced because a fraction of cells is still finishing DNA replication.This might be an explanation why the V. natriegens synSC1.0 cells have a shorter lag phase in minimal media (Figure S2).All plots were generated based on the BED files generated with Repliscope 6 .Data was normalized and plotted using R for details see Material and Methods.Data is available in Supplementary Data S2.The same samples were used to analyze the relative protein abundances (Figure 3C).

Figure S7 | Growth curves of replicates for replication pattern and shotgun proteomics samples.
Growth curve data of replicates of (A) the parental strain and (B) the synSC1.0strain.The color of each replicate is identical in both plots and visualized in purple, blue, green and orange.Replicates one to three are used for replication pattern analysis (Figure 3A-B and S6) and replicates one to four are used for shotgun proteomics (Figure 3C and S8).OD values are plotted to a log10 axis.Exponential growth phase samples for replication pattern analysis and shotgun proteomics were harvested at OD600 ≈ 0.3.Stationary phase samples for replication pattern analysis at after 12 hours at OD600 ≈ 10.

Figure S1 |
Figure S1 | Detailed annotation of genomic regions visualized in Figure 1C.Annotations are performed according to GenBank files CP009977 (chr1) and CP009978 (chr2) with their respective gene annotations.Purple and blue colors match the homology regions depicted in Figure 1A for the fusion of the two chromosomes.Red colors indicate the deletion of the ori2 region consisting of the parAB operon (PN96_16270 and PN96_16265) and rctB (PN96_16270).The scale of the genomic region is 10 kb.

Figure S2 |
Figure S2| Comparative growth assay parental strain and synSC1.0 in M9 media supplemented with 20.5 g/L NaCl and 0.4% glucose.(A) Doubling times are determined to be 22 min 46 s (+/-43.8s) and 23 min 39 s (+/-24.6 s) for the parental and synSC1.0strain, respectively.The difference in growth rate is 4.1%.E. coli was used as a control under the same conditions and doubling time was determined to be 1 h 40 min 12 s (+/-3 min 20.8 s).However, the conditions presumably cause high salt stress for E. coli.Student's t-test was applied to determine the significance; *: p< 0.01, **: p<0.001, n.s.not significant.Experiments were performed biological quadruplicates each with technical triplicates.(B) Growth curve of V. natriegens parental (left panel) and synSC1.0(right panel).We observed no extended lag-phase for synSC1.0 in repeated experiments from dense overnight cultures.Previously, we observed shorter lag-phase for synSC1.0which may be explained by at least a fraction of cells still replication (see FigureS6D-E for replication pattern details).
Figure S2| Comparative growth assay parental strain and synSC1.0 in M9 media supplemented with 20.5 g/L NaCl and 0.4% glucose.(A) Doubling times are determined to be 22 min 46 s (+/-43.8s) and 23 min 39 s (+/-24.6 s) for the parental and synSC1.0strain, respectively.The difference in growth rate is 4.1%.E. coli was used as a control under the same conditions and doubling time was determined to be 1 h 40 min 12 s (+/-3 min 20.8 s).However, the conditions presumably cause high salt stress for E. coli.Student's t-test was applied to determine the significance; *: p< 0.01, **: p<0.001, n.s.not significant.Experiments were performed biological quadruplicates each with technical triplicates.(B) Growth curve of V. natriegens parental (left panel) and synSC1.0(right panel).We observed no extended lag-phase for synSC1.0 in repeated experiments from dense overnight cultures.Previously, we observed shorter lag-phase for synSC1.0which may be explained by at least a fraction of cells still replication (see FigureS6D-E for replication pattern details).

Figure S3 |
Figure S3 | MIC assay for ciprofloxacin with smaller dilution steps to compare the parental and synSC1.0strain.No differences are observed in four biological replicates.A representative scan is shown.

Figure S4 |
Figure S4 | Comparative fluctuation assay of the parental strain and synSC1.0.No drastic differences were observed between the two strains which is consistent with the MIC test and other characterization experiments (see Figure 2).Experiments were performed in biological quadruplicates, CFU values are provided in Table S1.Error bars indicate standard deviation.

Figure S5 |
Figure S5 | Elongated cell phenotype observed for V. natriegens under rapid growth conditions.Elongated cells were sporadic observed in both the parental and synSC1.0strain.Representative image for the parental (left panel) and synSC1.0(right panel) are shown.The observed phenotype was more frequent for synSC1.0(cf.Figure2F).It may be the result of reduced chromosome dimer resolution fidelity previously observed in V. cholerae1 .More severe phenotypes affecting the whole cell population were described earlier for a natural isolate with fused chromosomes of V. cholerae but are not observed for synSC1.0 2 .However, recent literature indicates that there are potential challenges with cell division licensing in MCH1 presumable based on nucleoid occlusion system due to misplacement of the SlmA protein3,4 .Scale bar 2 µm.
Figure S5 | Elongated cell phenotype observed for V. natriegens under rapid growth conditions.Elongated cells were sporadic observed in both the parental and synSC1.0strain.Representative image for the parental (left panel) and synSC1.0(right panel) are shown.The observed phenotype was more frequent for synSC1.0(cf.Figure2F).It may be the result of reduced chromosome dimer resolution fidelity previously observed in V. cholerae1 .More severe phenotypes affecting the whole cell population were described earlier for a natural isolate with fused chromosomes of V. cholerae but are not observed for synSC1.0 2 .However, recent literature indicates that there are potential challenges with cell division licensing in MCH1 presumable based on nucleoid occlusion system due to misplacement of the SlmA protein3,4 .Scale bar 2 µm.

Figure S8 |
Figure S8 | Volcano plots of shotgun proteome analysis.(A) Visualizes the differential protein abundance for chr1 (red) and chr2 (blue) encoded gene products.(B) and (C) visualize chr1 and chr2 separately.The results show that the altered marker frequency based on the chromosomal fusion leads to a higher abundance of proteins encoded in chr1 and to a reduction of chr2 encoded proteins.WT = parental strain.

Figure S9 |
Figure S9 | Comparison of protein intensity ratios between synSC1.0 and the parental strain for genes required for rapid growth and all other genes.Classification of genes and associated proteins was based on the CRISPRi screen by Lee et al. 2019 7 .Data for genes with a significant beta score in LB3 are shown in the left boxplot (n = 584).Data for all other genes boxplot are shown in the right boxplot (n = 2276).P-value was calculated with a two-sample t-test.

Figure S10 |
Figure S10 | Volcano plots of proteome data based on selected KEGG pathway enrichments.No drastic alterations of protein levels can be observed for core pathways of V. natriegens synSC1.0.Strikingly, DNA replication and repair does not show a global alteration matching the data of the other synSC1.0characterization experiments.WT = parental strain.